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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil

Definitions

• the present invention concerns a novel compound having neuritogenic and antibacterial activities which is produced by a novel strain of Microtetraspora.
• Substances active against microorganisms have many beneficial uses. These uses are in fields such as human health care, veterinary science, and animal husbandry. Antimicrobial agents can have many desirable effects such as preventing or curing disease and promoting the growth of animals.
• New antimicrobial agents are needed for several reasons; these include intolerance of the subject to be treated to known antimicrobials, and the development of strains resistant to known antimicrobials. Therefore, characterization of any previously known microorganisms which produce new antimicrobial agents is highly desirable.
• Nerve growth factor is a protein known to be essential for the development and maintenance of certain sympathetic and sensory neurons in the peripheral nervous system (Thoenen, et al. Physiol. Rev. 60:1284-1335, 1980). Recent evidence also suggests important functions of NGF in the cholinergic neurons of the basal forebrain in the central nervous system (Whittemore, et al. Brain Res. Rev. 12:439-464, 1987). It has further been reported that in aged rodents, impairments in learning and memory are related with age-dependent decline in the forebrain of cholinergic functions (Goelet, et al.
• lactacystin is a microbial metabolite produced by a Streptomyces strain (Omura, et al. J. Antibiotics 44:113-116, 1991).
• the present invention is based on the discovery of a new microorganism strain which produces a compound which has been found to have antibacterial activity and neuritogenic activity.
• the compound of the invention has the formula ##STR1##
• the compound of the invention will be referred to herein as compound "BU-4514N" which term also includes pharmaceutically acceptable salts of the compound.
• the present invention is also directed to a pharmaceutical composition comprising compound BU-4514N together with a pharmaceutically acceptable carrier.
• Compound BU-4514N has been found to be produced by a microorganism identified as belonging to the genus Microtetraspora and designated as species T689-92.
• the present invention is also directed to a biologically pure culture of Microtetraspora species T689-92. It is also contemplated that mutants and variants of Microtetraspora species T689-92 are also within the scope of the present invention, whether created by conventional physical or chemical means or by recombinant genetic engineering techniques.
• the present invention also includes a process for producing a neuritogenic or antibacterial compound comprising cultivating under aerobic conditions Microtetraspora species T689-92 or a mutant or variant thereof in a suitable culture medium containing a carbon source and a nitrogen source at a pH and temperature and for a time sufficient for production of said compound.
• the present invention is directed to a method for controlling bacteria which comprises applying to said bacteria or habitat thereof an effective amount of compound BU-4514N.
• the present invention is directed to a method for enhancing the effect of nerve growth factor in a patient in need thereof which comprises administering to said patient an effective amount of compound BU-4514N.
• the present invention is directed to a method for treating a neurodegenerative disease in a patient in need thereof which comprises administering to said patient an effective amount of compound BU-4514N.
• FIG. 1- IR spectrum of BU-4514N.
• FIG. 2-- 1 H NMR spectrum of BU-4514N is a diagrammatic representation of BU-4514N.
• Microtetraspora species T689-92 was isolated from a soil sample collected in Andhra Pradesh, India. This organism has been deposited on Jan. 31, 1992, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852, U.S.A., under Accession Number ATCC-55291.
• ATCC American Type Culture Collection
• the present invention is not limited to use of the particular Strain T689-92 or to organisms fully answering the description contained herein. It is especially intended to include other BU-4514N producing strains or mutants or variants of said organisms which can be produced from the described organism by known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, and the like; or through the use of recombinant genetic engineering techniques.
• the compound of the present invention is produced by cultivating Microtetraspora species T689-92, or a mutant or a variant thereof, in a conventional aqueous medium.
• the organism is grown in a nutrient medium containing known nutritional sources for actinomycetes, i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors.
• Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotic, although for production of limited amounts, surface cultures and bottles may also be used.
• the general procedures used for the cultivation of other actinomycetes are applicable to the present invention.
• the nutrient medium should contain an appropriate assimilable carbon source such as sucrose, xylose, inositol, mannitol, fructose, or rhamnose.
• an appropriate assimilable carbon source such as sucrose, xylose, inositol, mannitol, fructose, or rhamnose.
• nitrogen sources ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. may be used or organic nitrogen sources such as peptone, fish meat extract, yeast extract, corn steep liquor, soybean powder, cotton seed flour, etc. may be used, or any combination thereof.
• nutrient inorganic salts may also be added if necessary nutrient inorganic salts to provide sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron and the like.
• optimum production of BU-4514N is obtained in shake flasks after an appropriate incubation period. Aeration in shake flasks is achieved by agitation, e.g. shaking on a rotary shaker. If fermentation is to be carried out in tank fermentors, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture from a slant culture of a lyophilized culture of the organism.
• BU-4514N After obtaining an active inoculum in this manner, it is aseptically transferred to the fermentation tank medium. Agitation in the tank fermentor is provided by stirring and aeration may be achieved by injection of air or oxygen into the agitated mixture. Production of BU-4514N may be monitored using chromatographic or spectroscopic techniques, or by a conventional biological assay.
• Preferred culture conditions include a pH of about 6 to about 8 and temperature of about 18° C. to about 44° C., and an incubation period of about 3 days to about 8 days. More preferred conditions include a pH of about 7, a temperature of about 31° C. to about 42° C., and an incubation period of about 4 days to about 6 days.
• the fermented whole broth can be extracted by contact with a suitable organic solvent such as n-butanol, n-butyl acetate or 4-methyl-2-pentanone, preferably under agitation.
• a suitable organic solvent such as n-butanol, n-butyl acetate or 4-methyl-2-pentanone, preferably under agitation.
• the organic layer can then be separated, e.g. by centrifugation followed by removal of the solvent, e g. by evaporation to dryness, preferably under vacuum.
• the resulting residue can then optionally be reconstituted (e.g. in a water ethyl acetate mixture) and re-extracted with a suitable organic solvent.
• the active compound can be further purified/isolated by use of standard techniques such as chromatography, particularly column chromatography, optionally followed by further purification, e.g. by use of reverse phase chromatography.
• standard techniques such as chromatography, particularly column chromatography, optionally followed by further purification, e.g. by use of reverse phase chromatography.
• HPLC high performance liquid chromatography
• the pharmaceutically acceptable acid addition salts those salts in which the anion does not contribute significantly to toxicity or pharmacological activity of their organic cation, are preferred.
• the acid addition salts are obtained either by reaction of the active compound with an organic or inorganic acid, preferably by contact in solution, or by any of the standard methods detailed in the literature and available to a practitioner skilled in the art.
• salts of the compound of the invention are illustratively hydrochloric, sulfuric, phosphoric, acetic, benzoic, citric, malonic, salicylic, malic, fumaric, oxalic, succinic, tartaric, lactic, gluconic, ascorbic, maleic, aspartic, benzenesulfonic, methane and ethanesulfonic, hydroxymethane- and hydroxyethanesulfonic. (See, for example, "Pharmaceutical Salts," J. Pharm. Sci. 66(1):1-19 (1977))
• Control refers to antimicrobial activity such as the suppression, inhibition, kill, stasis, or destruction of microorganisms, or any interference with the growth of microorganisms which results in a slower growth rate.
• effective amount when used in conjunction with the antimicrobial method of the present invention refers to that amount of compound BU-4514N sufficient to result in control of microorganisms.
• Compound BU-4514N can be used in a wide variety of applications in which control of microorganisms is desired.
• the compound is active against pathogenic and non-pathogenic bacteria which may be resistant to widely used known antibiotics. Because of this activity, BU-4514N can be used as therapeutic agents and administered to a patient in need of antibacterial treatment either orally, parenterally, or topically.
• treatment refers to amelioration, cure, or prevention of the appropriate disease or infection.
• Compound BU-4514N or combinations containing the same can also be used as disinfectants, for example, to disinfect objects and instruments.
• the compound can be used as an antibacterial agent, for example, by contacting bacteria pests or their habitat with effective amounts sufficient to obtain control of many organisms.
• the compound of this invention can be incorporated into various products susceptible to microbial degradation in order to prevent such degradation of the products by the microorganisms.
• Compound BU-4514N is also useful for treating neurodegenerative diseases.
• compound BU-4514N is useful in treating senile cognitive decline and Alzheimer's disease.
• the compound is also useful in treating myasthenia gravis, tardive dyskinesia, and dementia associated with Down's syndrome or Parkinson's disease.
• This utility of compound BU-4514N in treating these neurodegenerative diseases is demonstrated using culture of PC12 rat pheochromocytoma cells which have been shown to respond to nerve growth factor (NGF) (Tischler and Greene, Nature 258:341-342, 1975).
• NGF nerve growth factor
• the PC12 rat pheochromocytoma cells respond to NGF by differentiating into sympathetic neuron-like cells. The cells cease dividing, extend processes resembling neurites, and synthesize increased levels of neurotransmitters and neurotransmitter receptors.
• Compound BU-4514N can be administered to a patient in need of neurodegenerative disease treatment either orally or parenterally.
• the amount of compound to be administered for either the antibacterial or neuritogenic method of the invention would depend in part on the age, weight, and general condition of the patient. Typically, a patient would be closely monitored by a physician who could determine if the dosage amount or regimen of compound being administered was effective and well tolerated.
• Compound BU-4514N would be administered for either antibacterial or neuritogenic method of the invention either alone or admixed with a pharmaceutically acceptable carrier.
• An effective unit dose of the compound for the neuritogenic method would be from about 5 to about 50 mg/kg of body weight of the patient with a daily dose ranging from about 15 to about 150 mg/kg of body weight of the patient.
• a typical effective unit dose of the compound given orally or parenterally would be from about 5 to about 150 mg/kg of body weight of the patient with a daily dose ranging from about 15 to about 450 mg/kg of body weight of the patient.
• inert, pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositories.
• a solid carrier can be one or more substances which may also act is diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
• the carrier is a finely divided solid which is in a mixture with the finely divided active component.
• the active compound is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
• Powders and tablets preferably contain between about 5% to about 50% by weight of the active ingredient.
• Suitable carriers are magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.
• preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it.
• cachets are also included.
• the compound may be incorporated into lotions, ointments, gels, creams, salves, tinctures, and the like.
• Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
• Liquid form preparations include solutions suitable for oral or parenteral administration; suspensions, or emulsions suitable for oral administration.
• Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, ethanol, or propylene glycol may be mentioned as examples of liquid preparations suitable for parenteral administration.
• Sterile solutions for injection or infusion may be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
• Aqueous solutions for oral administration can be prepared by dissolving the active compound in water and adding suitable flavorants, coloring agents, stabilizers, and thickening agents as desired.
• Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.
• the pharmaceutical preparation is in unit dosage form.
• the preparation is divided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, packeted tablets, capsules and powders in vials or ampoules.
• the unit dosage form can also be capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.
• Biomass for chemotaxonomic analysis were prepared by using lyophilized whole cells grown at 32° C. for 4 days with a rotary shaker in a liquid medium (glucose 1% and yeast extract 1%, pH 7.0). Cell wall analysis was performed by the methods of H. A. Lechevalier and M. P. Lechevalier (A critical evaluation of the genera of aerobic actinomycetes. In The Actinomycetales [H. Prauser, ed.], pp. 393-405. Jena, Gustav Fischer Verlag, 1970) as modified by Staneck and Roberts (J. L. Staneck and G. D. Roberts: Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl. Microbiol.
• Phospholipid and mycolate composition were determined after Lechevalier (Lechevalier, et al: Chemotaxonomy of aerobic actinomycetes: Phospholipid composition. Biochem. Syst. Ecol. 5:249-260, 1977) and Minnikin (Minnikin, et al: Differentiation of Mycobacterium, Nocardia and related taxa by thin-layer chromatographic analysis of whole-organism methanolysates. J. Gen. Microbiol. 88:200-204, 1975), respectively. Menaquinone was analyzed by the procedure of Collins et al (A note on the separation of natural mixtures of bacterial menaquinones using reverse-phase thin-layer chromatography.
• Substrate mycelia were well developed, extensively branched and did not fragment into short elements or form spores. Rudimentary and retarded aerial mycelia were only produced on both yeast starch agar and Gauze's I agar, but no sporulating aerial mycelium was observed by scanning electron microscope. Therefore, several attempts to yield sporulating aerial mycelia have been made, without success.
• Strain T689-92 formed good vegetative growth on the media used in this study. Aerial mycelia were only produced on yeast starch agar, Gauze's I agar and maltose-Bennett's agar. They were rudiment without sporulation and were powdery with pale reddish yellow to grayish yellow. The color of vegetative mycelia and reverse side of colony ranged from pale yellow to reddish brown or dull yellow. No diffusible pigment was produced. The macroscopic properties of strain T689-92 on various agar media were summarized in Table 1.
• Strain T689-92 was propagated on rB agar slant composed of soluble starch (Nichiden Kagaku Co.) 0.5%, glucose 0.5%, meat extract (Mikuni Chemical Industries Co., Ltd.) 0.1%, yeast extract (Oliental Yeast Co.) 0.1%, NZ-case (Humko Sheffield Chemical Co.) 0.2%, NaCl 0.2%, CaCO 3 0.1% and agar (Junsei Chemical Co.) 1.6%, for 7 days at 28° C. A portion of the mature agar slant was inoculated into 100 ml of seed medium in a 500-ml Erlenmeyer flask and incubated for 4 days at 28° C.
• the vegetative medium was composed of soluble starch 2%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, fish meal D30X (Banyu Nutrient) 0.5% and CaCO 3 0.3%.
• the resulting vegetative mycelia were spun down gently (3,000 rpm, 15 min. 4° C.) and resuspended with a half volume of 20% aq. glycerol solution and then stocked in -80° C.
• a portion (0.3 ml) of vegetative mycelia of the producing strain was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of a seed medium composed of sucrose 3%, fish meal (Hokuyo Suisan Co.) 2%, soybean meal (Nikko Seiyu Co.) 0.5%, peptone (Nihon Seiyaku Co.) 0.2% and CaCO 3 0.6%.
• the pH of the medium was adjusted to 7.0 before autoclaving.
• the seed was incubated at 28° C. for 4 days on a rotary shaker operating at 200 rpm.
• a 5-ml portion of the seed culture was transferred into a 500-ml Erlenmeyer flask containing 100 ml of a production medium having the same composition as the seed medium. Fermentation was carried out for 6 days under the same conditions as those for the seed culture. The antibiotic activity was determined by the nerve growth factor mimic activity.
• the harvested whole broth (9.0 L) was extracted by stirring with n-butanol (4.5 L).
• the organic layer was separated by the aid of a Sharples centrifuge (Kokusan No. 4A) and evaporated to dryness under reduced pressure.
• the residue was dissolved in a mixture of ethyl acetate and water (1:1, 1.5 L). After stirring 15 minutes, aqueous layer was separated and re-extracted with n-butanol (750 ml).
• the n-butanol extract was evaporated under reduced pressure to give 2.38 g of crude product which was applied on a column of silica gel (Wakogel C-200, 600 ml).
• the column was developed with CH 2 Cl 2 :EtOH (1:1, 1 L) and CH 2 Cl 2 : EtOH: conc. NH 4 OH:H 2 O (10:10:1:1, 2.2 L), successively.
• the eluate was collected in 20-ml fractions and each fraction was monitored by TLC (SiO 2 :CH 2 Cl 2 :EtOH:conc. NH 4 OH:H 2 O (4:7:1:1) and nerve growth factor mimic activity.
• the active fractions were combined and evaporated to dryness to give 617 mg of light brown powder.
• This sample was chromatographed on a reversed phase silica gel column (YMC GEL ODS A60; Yamamura Chemica Lab., 700 ml) using aqueous acetonitrile (40%-80%) containing 0.1% of trifluoroacetic acid as an eluant.
• the eluate was monitored by TLC (SiO 2 :CH 2 Cl 2 :EtOH:conc. NH 4 OH:H 2 O (4:7:1:1) and the activity.
• the active fractions were combined and evaporated under reduced pressure to afford 603 mg of white amorphous powder.
• BU-4514N was obtained as colorless fine needles and its physico-chemical properties are summarized in Table 5.
• BU-4514N is practically insoluble in water and ordinary organic solvents, such as chloroform, ethyl acetate and benzene. But it is soluble in acidic water, dimethylsulfoxide and methanol. It showed positive responses to iodine, ferric chloride and ninhydrin reagents on TLC plate and negative to Sakaguchi reaction.
• the UV spectrum of BU-4514N exhibited maxima at 249 nm and 286 nm in methanol.
• the IR and 1 H NMR spectra of BU-4514N are shown in FIG. 1 and FIG. 2, respectively.
• the 13 C NMR spectrum of BU-4514N revealed 27 carbon signals (Table 6). The molecular formula of this compound was established to be C 27 H 42 N 2 O 5 by FAB-MS spectrum and microanalysis
• PC 12R cells derived from PC 12 cells have a sensitivity as high as 1 ng/ml to NGF, and were cultured in Opti-MEM medium (GIBCO) containing 5% heat-inactivated fetal bovine serum (GIBCO) and 50 ⁇ g/ml of amikacin (Bristol-Myers Squibb), in a humidified atmosphere of 5% CO 2 and 95% air at 37° C. to a cell density of approximately 1 ⁇ 10 8 cells per 175 cm 2 tissue Culture flask (Lux).
• Opti-MEM medium containing 5% heat-inactivated fetal bovine serum (GIBCO) and 50 ⁇ g/ml of amikacin (Bristol-Myers Squibb)
• the PC 12R cell density was adjusted to 1 ⁇ 10 5 cells/ml and 180 ⁇ l aliquots of the cell suspension were added to assay wells in a 96 well microtiter plate (Suimitomo).
• 0.1 ng/ml NGF Biomedical Technologies Inc. #BT-206 was first added to a well.
• 5.0 ng/ml NGF was added to a well.
• PC 12R cells without NGF were also prepared. Cultures were pre-incubated for 3 hours at 37° C. in a 5% CO 2 -95% air environment.
• the stained cells were observed under a phase-contrast microscope connected with a video camera-CRT system.
• PC 12R cells were magnified 500 times on the CRT monitor.
• the lengths of spikes of the negative control cells were measured in order to establish the standard length of a spike of the PC 12R cells.
• Tentatively positive is a test cell which contains the same or a higher number than the positive control of neurites which are over 10 mm long as composed with the standard length.
• the MIC's of BU-4514N against various test microorganisms were determined by the serial 2-fold dilution method after overnight incubation at 32° C. Nutrient agar medium (Difco) was used for Gram-positive and Gram-negative bacteria.
• BU-4514N significantly induces the neurite outgrowth in number and length at a concentration range from 12.5 to 6.3 ⁇ g/ml, compared to the negative control cells (Table 7).

Abstract

Description

______________________________________                                    
NGF Mimic Compounds Reported                                              
Compound   Note        Reference                                          
______________________________________                                    
Catecholamines                                                            
           NGF inducer Y. Furukawa, et al. J. Biol.                       
                       Chem. 261 6039-6047, 1986                          
                       S. Furukawa, et al. Biochem.                       
                       Biophys. Res. Commun. 147                          
                       1048-1054, 1987                                    
Aminoalkylester                                                           
           NGF inducer S. Furukawa, et al. Japan                          
derivs.                Open-Laid Patent: 2-172955                         
Gangliosides                                                              
           NGF-like    S. Tsuji, et al. J. Biochem. 94                    
                       303-306, 1983                                      
                       Cuello, et al. Proc. Natl.                         
                       Acad. Sci. 86 2056-2060,                           
                       1989                                               
                       Wu, et al., J. Neurochem. 56                       
                       95-104, 1991                                       
                       Tsuji, et al. J. Neurochem.                        
                       50 414-423, 1988                                   
Sialyl compounds                                                          
           NGF-like    Tsuji, et al. J. Neurochem.                        
                       50 414-423, 1988                                   
Saponins   Neuronotrophic                                                 
                       Himi, et al. Chem. Pharm.                          
           activity    Bull. 37 481-484, 1989                             
1,25-Dihydroxy-                                                           
           NGF inducer Wion, et al. J. Neurosci.                          
vitamin D.sub.3        Res. 28 110-114, 1991                              
Adenosine  Neuritogenesis                                                 
                       Huffaker, et al. J. Cell                           
           inducer     Physiol. 120 188-196 1984                          
                       Guroff, et al. J. Neurochem.                       
                       37 1431-1439, 1981                                 
Staurosporine                                                             
           Differentiation                                                
                       Morioka, et al. Agric. Biol.                       
           inducer     Chem. 49 1959-1963, 1985                           
Lactacystin                                                               
           Neuritogenesis                                                 
                       Omura, et al. J. Antibiotics                       
           inducer     44 113-116, 1991                                   
Peptide hormones                                                          
           NGF inducer Wion, et al. FEBS Lett. 189                        
                       37-41, 1985                                        
Retinoic acid                                                             
           NGF inducer Wion, et al. Biochem.                              
                       Biophys. Res. Commun. 149                          
                       510-514, 1987                                      
Phorbol 12-myri-                                                          
           NGF inducer Wion, et al. FEBS Lett. 262                        
state 13-acetate       42-44, 1990                                        
Dibutyryl-cyclic                                                          
           Neuritogenesis                                                 
                       Prasad, et al. Cancer 36 1338                      
AMP        inducer     1975                                               
______________________________________                                    

                                  TABLE 1                                 
__________________________________________________________________________
Cultural characteristics of strain T689-92                                
             Vegetative                            Diffusible             
Medium       mycelium      Reverse Side Aerial Mycelium                   
                                                   Pigment                
__________________________________________________________________________
Sucrose nitrate agar                                                      
             Pale yellow (128)                                            
                           Soft reddish yellow (146)                      
                                        None       None                   
(Waksman med. 1)                                                          
Glycerol nitrate agar                                                     
             Pale yellow (128)                                            
                           Soft yellow (147)                              
                                        None       None                   
Glucose asparagine agar                                                   
             Soft reddish yellow (146)                                    
                           Soft reddish yellow (146)                      
                                        None       None                   
(Waksman med. 2)                                                          
Yeast extract-malt extract                                                
             Grayish brown (118)-                                         
                           Yellowish brown (99)                           
                                        None       None                   
agar (ISP med. 2)                                                         
             dark yellowish brown (105)                                   
Oatmeal agar Soft reddish brown (146)                                     
                           Soft reddish brown (146)                       
                                        None       None                   
(ISP med. 3)                                                              
Inorganic salts-starch                                                    
             Gold (162)    Soft reddish yellow (146)                      
                                        None       None                   
agar (ISP med. 4)                                                         
Glycerol asparagine agar                                                  
             Dull yellow (150)                                            
                           Dull yellow (150)                              
                                        None       None                   
(ISP med. 5)                                                              
Tyrosine agar                                                             
             Dull yellow (150)                                            
                           Dull yellow (150)                              
                                        None       None                   
(ISP med. 7)                                                              
Nutrient agar                                                             
             Gold (161)    Gold (161)   None       None                   
(Waksman med. 14)                                                         
Yeast starch agar                                                         
             Yellowish brown (98)                                         
                           Grayish brown (118)                            
                                        Pale reddish yellow               
                                                   None                   
                                        (130), Powdery, Thin              
Gauze's agar Yellowish brown (99)                                         
                           Yellowish brown (99)                           
                                        Grayish yellow (155),             
                                                   None                   
                                        Powdery, Thin                     
Oatmeal-yeast extract agar                                                
             Soft reddish yellow (146)                                    
                           Soft reddish yellow (146)                      
                                        None       None                   
Bennett's agar                                                            
             Yellowish brown (99)                                         
                           Yellowish brown (99)                           
                                        None       None                   
(Waksman med. 30)                                                         
Maltose-Bennett's agar                                                    
             Soft reddish yellow (146)                                    
                           Brownish gold (160)                            
                                        Pale reddish yellow               
                                                   None                   
                                        (130) Powdery, Thin               
__________________________________________________________________________

              TABLE 2                                                     
______________________________________                                    
Physiological Characteristics of Strain T689-92                           
Test                   Results                                            
______________________________________                                    
Starch hydrolysis (On ISP med. No. 4)                                     
                       Positive                                           
Nitrate reduction (Difco, nitrate broth)                                  
                       Positive                                           
Milk (Difco, 10% skimmed milk)                                            
Coagulation            Positive                                           
Peptonization          Positive                                           
Cellulose decomposition                                                   
                       Negative                                           
(sucrose nitrate solution with a                                          
paper strip as the sole carbon source)                                    
Gelatin liquefaction                                                      
On plain gelatin       Negative                                           
On glucose peptone gelatin                                                
                       Negative                                           
Melanin formation (On ISP med. No. 7)                                     
                       Negative                                           
Temperature range for growth (°C.)                                 
                       18-44                                              
Optimum temperature (°C.)                                          
                       31-42                                              
(On Yeast starch agar)                                                    
pH range for growth    6-8                                                
Optimum pH             7                                                  
(On Trypticase soy broth, BBL)                                            
______________________________________                                    

              TABLE 3                                                     
______________________________________                                    
Utilization of Carbon Sources by Strain T689-92                           
Carbon Source   Growth                                                    
______________________________________                                    
D-Glucose       ++                                                        
L-Arabinose     ±                                                      
D-Xylose        +                                                         
Inositol        ++                                                        
Mannitol        +                                                         
D-Fructose      +                                                         
L-Rhamnose      +                                                         
Sucrose         +                                                         
Raffinose       -                                                         
______________________________________                                    
 -: Negative                                                              
 ±: Doubtful                                                           
 +: Weak positive                                                         
 ++: Strong positive                                                      
 (ISP med. No. 9, 37° C. for 21 days)                              

                                  TABLE 4                                 
__________________________________________________________________________
Fatty Acid Composition of Strain T689-92                                  
Fatty Acid Composition (%)                                                
Straight Chain                                                            
            Branched Chain Unsaturated Chain                              
__________________________________________________________________________
14:0                                                                      
   15:0                                                                   
      16:0                                                                
         17:0                                                             
            i-14                                                          
               i-15                                                       
                  i-16                                                    
                     i-18                                                 
                        a-17                                              
                           15:1.sup.9                                     
                              i-16:1                                      
                                 16:1.sup.9                               
                                    i-18:1.sup.9                          
                                        10Me16                            
                                             10Me17                       
1  2  2  3  1  3  26 1  5  1  4  2  1   1    21                           
10Me18                                                                    
     2OH15                                                                
         i-2OH16                                                          
              2OH16                                                       
                  i-2OH17                                                 
                       OH17:0                                             
4    1   10   2   1    2                                                  
__________________________________________________________________________

              TABLE 5                                                     
______________________________________                                    
Physico-chemical Properties of BU-4514N                                   
______________________________________                                    
Nature          colorless fine needles                                    
Melting point   185-186° C.                                        
Optical rotation                                                          
                [α].sup.25 -140 ± 1° (c. 0.5, 0.1N HCl)   
Mass (FAB-MS) m/z                                                         
                475 (M + H).sup.+                                         
Molecular formula                                                         
                C.sub.27 H.sub.42 N.sub.2 O.sub.5                         
Elemental analysis                                                        
Calcd for C.sub.27 H.sub.42 N.sub.2 O.sub.5 3/4H.sub.2 O                  
                C 66.43, H 8.98, N 5.74                                   
Found           C 66.21, H 8.94, N 5.66                                   
UV λ.sub.max nm(ε)                                         
MeOH            286(8530), 251(4360), 204(6300)                           
0.01N HCl/MeOH  286(9000), 254sh(4320), 204(6540)                         
0.01N NaOH/MeOH 287(8200), 248(9200), 205(8440)                           
IR ν.sub.max (KBr)                                                     
                3350, 2970, 2930, 1650, 1565, 1530                        
                1445, 1240, 1110, 1020, 995 cm.sup.-1                     
______________________________________                                    

              TABLE 6                                                     
______________________________________                                    
.sup.13 C and .sup.1 H NMR Spectral Data*.sup.1 for BU-4514N              
 ##STR3##                                                                 
Carbon                                                                    
No.    δ.sub.C (multiplicity)                                       
                      δ.sub.H (multiplicity)                        
______________________________________                                    
1      179.10(s)                                                          
2      104.20(s)                                                          
3      204.13(s)                                                          
4      52.34(s)                                                           
5      41.92(d)       1.55(m)                                             
6      29.62(t)       1.01(m), 1.84(m)                                    
7      37.72(t)       1.06(m), 1.74(m)                                    
8      35.34(d)       1.46(m)                                             
9      44.27(t)       0.76(q, J=12.4Hz), 1.76(m)                          
10     41.36(d)       1.78(m)                                             
11     126.10(d)      5.07(brs)                                           
12     137.56(s)                                                          
13     43.64(d)       2.57(br)                                            
14     42.90(t)       1.32(m), 1.53(m)                                    
15     78.71(d)       3.35(m)                                             
16     23.78(q)       1.04(d, J=5.98Hz)                                   
17     193.21(s)                                                          
18     51.71(t)       3.88(d, J=18.81Hz), 3.92(d,                         
                      J=18.81Hz)                                          
19     15.65(q)       13.4(s)                                             
20     24.01(q)       0.89(d, J=6.84Hz)                                   
21     23.70(q)       1.69(s)                                             
1'     100.49(d)      4.79(brs)                                           
2'     24.93(t)       1.66(m), 1.82(m)                                    
3'     23.70(t)       1.86(m), 2.17(m)                                    
4'     51.04(d)       3.32(m)                                             
5'     65.34(d)       4.21(dq, J=6.84Hz & 1.28Hz)                         
6'     18.14(q)       1.13(d, J=6.84Hz)                                   
______________________________________                                    
 *.sup.1 in a mixture of CD.sub.3 OD/D.sub.2 O/4NDCl (1:1:0.08)           

              TABLE 7                                                     
______________________________________                                    
Neurite Outgrowth Activity of BU-4514N and NGF                            
                      No. of Neurites*/                                   
Compound                                                                  
        Concentration No. of Cells**                                      
                                   Ratio (%)                              
______________________________________                                    
4514N   12.5 μg/ml 244/1123     21.7                                   
         6.3 μg/ml 149/1570     9.5                                    
         3.1 μg/ml  25/1848     1.4                                    
         1.6 μg/ml  2/1611      0.12                                   
NGF      0.1 ng/ml     2/1565      0.13                                   
        (Running control)                                                 
          5 ng/ml      72/1053     6.8                                    
        (Positive control)                                                
          0 ng/ml      0/1298      0                                      
        (Negative control)                                                
______________________________________                                    
 *Total number of neurites over 10 mm long (observed on the monitor; sum o
 5 fields/well and 3 wells/concentration)                                 
 **Total number of cells (sum of 5 fields/well and 3 wells/concentration) 

              TABLE 8                                                     
______________________________________                                    
Antibacterial Activity of BU-4514N                                        
Test Organism         MIC (μg/ml)                                      
______________________________________                                    
S. aureus    FDA 209P JC-1                                                
                          1.6                                             
S. aureus    Smith        3.1                                             
S. aureus    A15036 (MRSA)                                                
                          3.1                                             
M. luteus    PCI 1001     1.6                                             
B. subtilis  PCI 219      0.8                                             
E. coli      Juhl A15119  >100                                            
E. coli      K12          >100                                            
E. coli      NIHJ JC-2    >100                                            
K. pneumoniae                                                             
             PCI 602      >100                                            
C. fleundii  GN 7391      >100                                            
S. typhi     901          >100                                            
P. aeruginosa                                                             
             A9843A       >100                                            
______________________________________                                    
 Medium: Nutrient Agar, pH 7.0                                            
 Inoculum size: 10.sup.5 cells/ml                                         
 Incubations: 32° C., 18 hr                                        

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  • 1992-09-25 US US07/951,976 patent/US5266588A/en not_active Expired - Lifetime

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